Neuronal nicotinic acetylcholine receptors (AChRs) will be studied using stably transfected QT-6 quail cell lines to express epitope-tagged chick neuronal AChR gene constructs under inducible promoters. a7 AChRs will be given special emphasis because of their unusual properties (they function as homomers, have high calcium permeability and are located extrasynaptically in ciliary ganglia). Also to be studied are combinations of a3, b4, a5 + b2 subunits (because AChRs of these types are found postsynaptically in ciliary ganglia). The cell lines will be used to analyze subunit interactions, assembly pathways, and functional properties. Surface AChRs will be quantitated using toxins and mAbs to the epitope tags. Function will be assayed by whole cell patch clamp recording. Assembly intermediates will be identified by pulse labeling and immune precipitation. Subunit composition will be analyzed by immunoprecipitation and immunoblot analysis using mAbs to subunits and tags. Distribution within the cells will be evaluated by confocal microscopy. Neuronal cell lines which express a7 will also be used for comparison. These studies should reveal important aspects of the cell and molecular biology of neuronal nicotinic receptors and are relevant to the role of nicotinic AChRs in nicotine addiction.